Project Summary/Abstract Human cytomegalovirus (HCMV) is a common opportunistic pathogen, responsible for a variety of diseases, particularly in immunocompromised patients and newborns. Drug resistance is a major concern in the treatment of HCMV diseases. Ganciclovir (GCV), the first-line drug against HCMV, targets HCMV DNA polymerase and thus, after prolonged GCV therapy, single or multiple mutations in HCMV DNA polymerase (Pol) that confer various levels of resistance to GCV can arise, leading to the failure of HCMV therapy. Therefore, the overall goal of this study -- to gain a better understanding of GCV resistance -- is highly health- related. Published work by others and our previous publications and preliminary data suggest that GCV, which is incorporated internally into DNA by certain GCV-resistant HCMV mutant polymerases, must be removed for viral viability, leading to the central hypothesis that host DNA repair may be important for the antiviral resistance of these mutants. To our knowledge, a relationship between DNA repair and antiviral drug resistance has not yet been studied. In this application, we explore this new concept, and propose two Aims to investigate it. Aim 1 is to determine the fate of incorporated GCV in cells infected with mutant HCMV. Following exposure of mutant virus-infected cells to radiolabeled GCV, the relative amounts of internally incorporated GCV in viral DNA at various times post-infection will be determined, which would allow us to see whether the internally incorporated GCV in mutant virus DNA is removed; i.e. repaired. Aim 2 is to test whether mutant viruses remain resistant to GCV in cells deficient in certain DNA repair pathways. DNA repair-deficient cells will be tested to see if the mutant virus becomes sensitive to GCV in these cells. If so, the repair protein that is deficient in the cells and the corresponding DNA repair pathway will be considered as the candidate mechanism for repair of incorporated GCV. To follow up this finding, siRNA or CRISPR/Cas methods will be used to knock down or knock out the repair protein in normal cells to see if a similar phenotype is obtained and the WT gene for the repair protein will be introduced into mutant cells, which should restore GCV resistance to the mutant virus. In parallel, the fate of incorporated GCV in mutant virus DNA will be assessed in repair- deficient cells, using the methods utilized in Aim 1. In summary, this proposal investigates a problem in antiviral drug resistance that is important clinically and mechanistically. Identification of the relationship between antiviral resistance and DNA repair would reveal a new paradigm in viral drug resistance, advance our understanding of virus-host interactions, and provide information that might aid in the development of a new clinical practice for the treatment of drug-resistant infections.